This project focuses on mitochondria as a central target of chemical-induced apoptosis. During the previous award period, we found that mitochondrial release of cytochrome c (cyt c) and caspase activation is a mechanism common to toxicity induced by oxidants and alkylating agents. In this proposed renewal, we have shifted emphasis to upstream events that control sensitivity of mitochondria to release of cyt c. Chemicals that modify thiols (oxidants, alkylating agents and heavy metals) are known to activate the opening of a high conductance permeability transition (PT) pore in mitochondria that causes release of cyt c. Thus, systems that maintain protein thiols and protect against activation of the PT pore are likely to determine sensitivity to chemical-induced apoptosis. Members of the thioredoxin (Trx) family reduce protein disulfides and sulfenic acids and, along with glutathione-dependent systems, are the major known systems for protection of protein thiols. Mitochondria contain a specific form of thioredoxin (mtTrx) and the purpose of this proposal is to determine whether this protein has a central function in protection of mitochondrial protein thiols and thereby in protection against chemical-induced toxicity. Little is known about human mtTrx expression, activity or function, but our preliminary data show that over-expression in a human cell line results in protection against oxidant-induced cell death. We propose biochemical and molecular studies of human mtTrx to fulfill the following aims: Aim 1 is to characterize expression of human mtTrx and investigate its inducibility. Aim 2 is to determine catalytic and redox properties of the purified protein. Aim 3 is to examine mechanisms of mtTrx protection against oxidant-induced apoptosis. Aim 4 is to determine whether the reactive thiol in mtTrx is a selective target for alkylation and whether this alkylation is associated with activation of mitochondria-mediated apoptosis. Aims 1 and 2 will substantially add to basic knowledge of human mtTrx and Aims 3 and 4 will define whether mtTrx has a central function in protection against chemical-induced toxicity.